In‐field detection and characterization of B/Victoria lineage deletion variant viruses causing early influenza activity and an outbreak in Louisiana, 2019

Abstract Background In 2019, the Louisiana Department of Health reported an early influenza B/Victoria (B/VIC) virus outbreak. Method As it was an atypically large outbreak, we deployed to Louisiana to investigate it using genomics and a triplex real‐time RT‐PCR assay to detect three antigenically distinct B/VIC lineage variant viruses. Results The investigation indicated that B/VIC V1A.3 subclade, containing a three amino acid deletion in the hemagglutinin and known to be antigenically distinct to the B/Colorado/06/2017 vaccine virus, was the most prevalent circulating virus within the specimens evaluated (86/88 in real‐time RT‐PCR). Conclusion This work underscores the value of portable platforms for rapid, onsite pathogen characterization.


| BACKGROUND
During the 2016-2017 influenza season, the influenza B (Inf B/Victoria (B/VIC) lineage variant viruses emerged with two or three amino acid (AA) deletions within the K 162 N 163 D 164 region of the hemagglutinin (HA) protein and rapidly spread worldwide. 1These deletion variant viruses are antigenically distinct from each other and from the progenitor B/VIC virus that lacked the deletions, resulting in four cocirculating subclades that were genetically and antigenically distinct during the timeframe of this outbreak: V1A (no deletion), V1A.1 In August 2019 in Louisiana, the proportion of healthcare visits for influenza-like illness (ILI) began to increase, primarily in children, and presented earlier than previous seasons. 4 S1).RNA from these 88 specimens was manually extracted onsite at the LDH using Akonni TruTip nucleic acid purification kit according to the previously established Mia protocol. 6

| VIC lineage deletion detection triplex realtime RT-PCR assay
The VIC DEL triplex rRT-PCR assay includes a single set of conserved amplification primers and three deletion-specific dual-labeled hydrolysis probes, including VIC 2_Del, Vic 3_Del, and Vic No_Del probes.Three probes, targeted on the deletion region of the HA gene of B/VIC viruses, were designed to specifically detect and differentiate B/VIC V1A.1, V1A.2/V1A.3,and the V1A genetic subclade (no deletion) (V1A-NoDel) viruses (Table S1 and Figure S1).The VIC DEL triplex rRT-PCR probe's fluorescence and quenchers labeling were described previously 3 (Table S1).

| In-field detection of influenza B/Victoria lineage deletion variant viruses
The VIC DEL triplex rRT-PCR assay was performed in-field using a portable, 4.5 lb real-time PCR instrument, the Quantabio "Q" portable qRT-PCR instrument (Beverly, MA, USA) that tests 48 samples in $100 min/run.The rRT-qPCR reactions setup and thermocycling conditions were previously described. 3

| Onsite and follow-up sequencing
Sequencing was performed as described previously 6 with some modifications.Here, InfB specific primers were used to amplify the RNA via SuperScript IV one-step RT-PCR.Raw fast5 read files were base called and demultiplexed with Guppy v.2.3.7 using default parameters on a MinIT GPU device (Oxford Nanopore Technologies, Oxford, UK).Reads were mapped and assembled into influenza genomes using IRMA v.0.6.7 with a MinION configuration module. 7urality consensus sequences for each segment were used in analyses.Samples were subsequently transferred to CDC in Atlanta where the RNA was re-amplified using SuperScript III based multisegment RT-PCR (MRT-PCR) followed by Illumina (Illumina, San Diego, CA, USA) sequencing and analysis by the Influenza Division's surveillance pipeline. 7e AA deletion and substitutions of V1A HA sequences were analyzed using the BioEdit program (http://www.mbio.ncsu.edu/bioedit/).Phylogenetic analysis was performed using Molecular Evolutionary Genetics Analysis software (version 7.0 http://megasoftware. net).The evolutionary history was inferred using the maximum likelihood method.The evolutionary distances were computed using the Tamura-Nei method. 8A B L E 1 In-field performance of B/VIC DEL triplex rRT-PCR assay.
One large pediatric healthcare facility in New Orleans (Facility A) reported 1268 B/VIC virus infections confirmed by the Louisiana Department of Health (LDH) with the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel Influenza B Lineage Genotyping Kit, 5 including 23 hospitalizations from July 31 to November 21, 2019.During this period, LDH reported one pediatric death associated with B/VIC virus infection. 4Due to the uncharacteristically large outbreak, rapid viral characterization was needed to determine if this was a known circulating virus or a new subclade.During November 2019, Louisiana declared a state of emergency after a cybersecurity attack on state government servers limited the connectivity of the LDH and their ability to perform genetic characterization.Our team deployed to the LDH with our mobile influenza analysis (Mia) next-generation sequencing platform 6 and our portable B/VIC DEL triplex rRT-PCR assay, both of which were not reliant on the LDH system, to determine the viral subclades.2 | MATERIALS AND METHODS 2.1 | Clinical specimens and in-field RNA extraction Sixty-five respiratory specimens submitted to the LDH and 23 specimens from patients hospitalized in Louisiana with InfB virus infection between July 31 and November 21, 2019, were included in the study.These 88 specimens were previously determined to be InfB and B/VIC positive via testing by the LDH with the CDC Influenza B Lineage Genotyping Kit 5 (Table

1
Evolutionary relationship of hemagglutinin gene among influenza B/Victoria V1A viruses.HA (coding sequence) phylogenetic tree with representative viruses of three subclades of V1A amino acid deletion viruses V1A.1 (2DEL), V1A.2 (3DEL), and V1A.3 (3DEL) indicated by the bars on the right.The quantityr and representative viruses from all identical HA sequences obtained from this study are shown in bold and italic face.The value in the parentheses indicates the quantity of identical sequences.The influenza B vaccine viruses are indicated with *.The bootstrap value (1000 replicates) of HA gene is shown next to the branches.